New sulfoxydes of the alkyl thiocolchiceine series



United States Patent 2,8il,554 Patented Oct. 29, 195? 2,811,554 NEW SULFOXYDES F THE ALK L THIOCOLCHICEINE 13 Georges Muller and Leon Velluz, Paris, France, assignors to UCLAF, Paris, France, a corporation of France No Drawing. Application May 3, 1955, Serial No. 505,831

Claims priority, application France May 14, 1954 6 Claims. (Cl. 260--562) 7 The present invention relates to new sulfoxide compounds and more particularly to new sulfoxides of the alkyl thiocolchiceine series and to a process of making same.

It is one object of the present invention to provide new sulfoxides of the alkyl thiocolchiceine series, said compounds being of considerable value in the industrial field, particularly for the modification of kariokynesis and the production of polyploids, said sulfoxides being considerably less toxic than colchicine and other active compounds derived from plants of the genus Colchicum.

Another object of the present invention is to provide a simple and effective process of producing said new sulfl oxides of the alkyl thiocolchiceine series.

Still another object of the present invention is to provide new and valuable preparations containing said new sulfoxides, said preparations being useful as antimitotic agents, for instance, in agriculture for the treatment of seeds and of cultivated soil to cause polyploidism.

Other objects of the present invention and advantageous features thereof will become apparent as the description proceeds.

The new sulfoxide compounds of the alkyl thiocolchiceine series according to the present invention correspond to the Formula I given hereinafter. They are obtained by oxidation of the alkyl mercapto group -SR2 of alkyl thiocolchiceine compounds of the Formula II, also given hereinafter. Said alkyl thiocolchiceine compounds are obtained as described in detail inour'copending application Serial No. 486,036, filed February 3, 1955, and entitled New Thio-Derivatives of Colchiceine Compounds and a Process of Making Same. According to the process of said application a colchiceine or isocolchiceine compound is mixed with an excess of an alkyl mercaptan, preferably in the presence of an acid catalyst, and the mixture is allowed to react until the corresponding alkyl thiocolchiceine compound is formed which is then isolated from the reaction mixture. It is also possible to condense the colchiceine compound with an alkali metal salt of the alkyl mercaptan.

R() RO- H 0 0- NELR] H3O O NH.R1

HaC O HxC) 0 i=0 i R: v i it; Formula I Formula'II In said formulas, R is hydrogen, an acyl group and espe-' rially a lower aliphatic or a mononuclear aromatic acyl 'group, or an alkyl radical, especially a lower alkyl radical; R1 is hydrogen, an acyl group and especially a lower aliphatic or a mononuclear aromatic acyl group, or an alkyl radical, said acyl group or said alkyl radical being the same as or different from the acyl group or alkyl radical in position R; and R2 is a substituted or non-substituted alkyl radical and especially a lower alkyl radical which may be substituted by a hydroxyl group.

The general Formula I is written in agreement with the established structure of colchicine having the Formula III given hereinafter in which R is -CH3, desmethyl colchicine having the Formula III, in which R is H (see Santavy and Reichstein, Helv. Chim. Acta, 1950, vol. 33, page 1606), and colchicoside having the Formula III, in which R is the glucosidyl residue -CsH11O5. The production of said colchicoside is described and claimed in copending application Serial No. 332,115, filed January 19, 1954, now U. S. Patent No. 2,734,014. Its relationship with desmethyl colchicine has been established.

HaCO NH.CO.CH3

| OCH3 Formula III Compounds of the present invention which are of particular interest are the sulfoxides of thiocolchicine (corresponding to Formula I wherein R is -CH3, R1 is -CO.CH3, and R2 is -CH3), of desmethyl thiocolchicine (corresponding to Formula I wherein R is H, R1 is --CO.CH3, and R2 is CH3) and of ethyl thiocolchiceine (corresponding to Formula I wherein R is CH3, R1 is CO.CH3, and R2 is C2H5).

From the physiological point of view these new sulfoxides have a considerably lower toxicity than previously used colchicum derivatives isolated from plants of the genus Colchicum, such as meadow saffron. For instance, the toxicity (lethal dose 50) which is 2 in the case of colchicine and 1 in the case of thiocolchicine, is as high as 35 to 75 in the case of the new sulfoxides of thiocolchicine according to the present invention.

The new compounds are used in the biological and in dustrial field for the same purposes for which colchicine is used, particularly for the modification of kariokynesis and the production of polyploids, and are administered either by spreading aqueous solutions or suspensions thereof on cultivated soil, or by treating seeds in undiluted form therewith or with solutions of said compounds in a suitable solvent or with dusting powders containing said compounds deposited on a suitable carrier.

The new sulfoxides exist in the form of their optical antipodes of the Formulas Ia and Ib given hereinafter in which the asymmetrical element is sulfur, as has been established from work carried out on other sulfoxides (see Phillips, J. Chem. Soc., 1925, vol. 127, page 2552; Harrison, Kenyon and Phillips, J. Chem. Soc., 1926, vol. 128, page 2079; Schmid and Karrer, Helv. Chim. Acta,

1948, vol. 31, page 1947). To the two isomers obtained" according to the present invention from alkyl thioeols;

chiceine compounds there can be attributed the D and the L configuration as indicated in the Formulas Ia and Ib.

RO- R H 0- NH. R1 H30 0 NH.R

0 0 2 R2 Formula Ia Formulalb In order to produce the sulfoxides according to the present invention, preferably an alkyl thiocolchiceine compound having the above given Formula II (R being hydrogen, an acyl radical, preferably of the aliphatic or aromatic series, or an alkyl radical, preferably a lower alkyl radical; R1 being hydrogen, an acyl radical, preferably of the aliphatic or aromatic series, or an alkyl radical, preferably ,a lower alkyl -radical,';R1 being equal to or different from R; and R2 being an unsubstituted orsubstituted alkyl radical) is treated with a suitable oxidizing agent capable of converting sulfides into sulfoxides (see, for instance, Gilman, Organic Chemistry, volume 1, pp. 870-71, 2nd edition, 1947). Best results are obtained with hydrogen peroxide having a concentration of about 30% or with peracids, for instance, perphthalic acid or perbenzoic acid. It is also possible to use solutions of hypochlorous acid (Javelle water). The oxidizing agents are added to solutions of the alkyl thiocolchiceine starting material in suitable organic solvents, preferably at a temperature between about 0 C. and about C. The optical antipodes of the resulting new sulfoxides, if de sired, are separated from each other and are purified by means of fractional crystallization or chromatography. The new sulfoxides can be reduced by means of sodium bisulfite to the starting materials.

The following examples serve to illustrate the present invention without, however, limiting the same thereto. In particular it is possible to oxidize all alkyl thiocolchiceine compounds having the structural Formula II, in which R, R1, and R have .the above given designation, provided they contain only groups and substituents which are not vaffected by oxidation conditions such as are encountered when proceeding according to the present invention. It is also possible to convert :any derivative having the structural Formula I, in which R, R1 and R2 have the above given designation, into other derivatives corresponding to the same general Formula I by replacing the substituents R and R1 by other suitable substituents. For instance,,if R1 is hydrogen, the amino group can be acylated or alkylated to convert it into an acylated or alkylated amino group. Likewise, if R is hydrogen, the phenol group can be esterified or etherified by reaction with acylating or alkylating agents. These reactions may be carried out before or after converting the alkyl thiocolchiceine compounds into the corresponding sulfoxides.

The melting points given inthe examples are determined on the Maquenne block and represent instantaneous melting points.

of thiocolchicine (Formulae la and lb, R=-CH3, R1=CO-CH3, R2=-CH3) added during 30 minutes, while keeping the temperature of the mixture at 0 C. The chloroform solution is then washed, dried, and evaporated to dryness. The

4 resulting material is crystallized from ethyl acetate and the resulting crude product is recovered; it is then -recrystallized twice from a mixture of ethyl acetate and ether (3:2). In this way 1.95 g. (38%) of L-sulfoxide of thiocolchicine (Formula Ia,

(0.5% concentration in chloroform) are obtained.

The product is obtained in the form of yellow, square leaves which are soluble in '100 parts by volume of ethyl acetate and in approximately 5 parts by volume of chloro form, but are insoluble in diethyl ether and in petroleum ether.

Analysis-Found: C%, 61.4; H%, 5.9; 0%, 21.9; N%, 2.9; 8%, 7.7. C22H25O6NS (431.49) requires C%, 61.2; H%, 5.8; 0%, 22.3; N%, 3.2; 8% 7.4.

By evaporating the mother liquors remaining after crystallization of the L-sulfoxide, there is obtained a residue weighing 3.5 g. which is taken up in 18 cc. of hot ethyl acetate. 2.1 g. of a fraction having [a] =176 (0.5% concentration in chloroform) are obtained by means of fractional crystallization. After adsorption on a neutral alumina column, elution with portions of 250 cc. of chloroform is effected. The first four fractions give, after evaporation of the solvent, 1.55 g. (30%) of D-sulfoxide thiocolchicine (Formula Ib,

(0.5% concentration in chloroform). The product is present in the form. of yellow needles which are soluble in parts byvolume of boiling ethyl acetate, in 5 parts by volume of chloroform and inv 20 parts by volume of cold acetone, but is insoluble .in diethyl ether and petroleum ether.

Analysis.Found: C%, 61.3; H%, 6.2; 0%, 22.5; N%, 2.9; 8%, 7.5. C22H25O6NS (431.49) requires C%, 61.2; H%, 5.8; 0%, 22.3; N%, 3.2; 8%, 7.4.

It is easily possible to demonstrate that the compounds produced according to this example are isomeric sul foxides by reducing the same to thiocolchicine. This is done, for instance, by heating on a boiling water bath a solution of 0.1 g. of the L- or the D-sulfoxide of thiocolchicine (Formula In or lb) in 1 cc. of ethanol with 3 "cc. of sodium'bisulfi'te solution (35 Baum), extracting the reaction mixture with chloroform, and washing, drying, and evaporating the chloroform extract to dryness. By crystallization from ethyl acetate 18 mg. of pure thiocolchicine are obtained.

Example 2.Pr0ducti0n of L- and D-sulfoxides of thiocolchicine (Formulae -Ia and lb, R=CH3, R1=CO-CH3, R2=-CH3) 415 mg. of thiocolchicine are dissolved in 8 cc. of methanol and 8 cc. of water are added thereto. 1 cc. of Javelle water containing one equivalent of active oxy gen is then added to the solution. An excess of solid sodium bicarbonate is introduced into this solution and then a further cc. of Iavelle water. The mixture is heated for 30 minutes at 50 C., cooled, extracted with chloroform, and washed with water. The extract is dried, the solvent is removed and the residue is subjected to fractional crystallization from ethyl acetate.

In this way the L-sulfoxide of thiocolchicine having a melting point of 264 C. [a] =505, is separated; this compound is identical with the one obtained according to Example 1. The yield is 24%, while the yield of melting point 250 C.,

. 5 Example 3.--Prduction of D- and L-sulfoxides of desmethyl thiocolchicine (Formulae Ia and lb, R=H, R1=COCH3, R2=--CH3) l g. of desmethyl thiocolchicine (Formula II, R=H, Ri=-CO--CH3, R2=--CH3) is dissolved in 200 cc. of chloroform and 15 cc. of a 3.4% solution of perphthalic acid in chloroform are added thereto over a period of one hour. The chloroform solution is washed with sodium bicarbonate and with water, dried, and distilled to dryness. Fractional crystallization from ethyl acetate yields to a first crop of crystals which, after recrystallization from a mixture of methanol and ether (1:1), yields 290 mg. (28%) of the D-sulfoxide of desmethyl thiocolchicine having a melting point of 265 C. and 300 C. and [a] =--145:10 (0.25% concentration in chloroform). I

The product is present in the form of yellow needles which are soluble in six volumes of hot methanol, soluble in 400 volumes of cold chloroform, but very sparingly soluble in ethyl acetate, and insoluble in water.

Analysis.--Found: C%, 60.1; H%, 5.7; N%, 3.1; S%, 7.7. C21H23O6NS (417.46) requires C%, 60.4; H%, 5.6; N%, 3.3; S%, 7.7. The mother liquors from the crystallization-of the first crop of crystals are evaporated to dryness and the resulting residue is taken up in 10 cc. of ethyl acetate. The resulting solution is allowed to crystallize, filtered with suction, dried, and the residue is dissolved in a mixture of equal parts of methanol and ether. In this way 270 mg. (26%) of L-sulfoxide of desmethyl thiocolchicine having a melting point of 260 C. and

(0.25% concentration in chloroform) are obtained. The product is present in the form of yellow pyramidal crystals which are soluble in six volumes of hot methanol and in 400 volumes of cold chloroform, but which are very sparingly soluble in ethyl acetate and insoluble in water.

Analysis. -Found: C%, 60.1; H%, 5.7; 0%, 23.1; N%, 3.2; S%, 7.8. C21H2306NS (417.46) requires C%, 60.4; H%, 5.5; 0%, 23.0; N%, 3.3; S%, 7.7.

The mother liquors from the crystallization of the two sulfoxides are subjected to chromatographic treatment consisting in first evaporating to dryness in a vacuum said mother liquors, dissolving the residue in 5 parts by volume of chloroform, and allowing the resulting solution to pass through a chromatographic column containing 50 parts by Weight of aluminum oxide for each part by weight of said residue. The two sulfoxides in said column are developed by means of benzene and are successively eluated by means of chloroform. In this manner, a further crop of crystals is obtained to bring up the total yield of D-sulfoxide of desmethyl thiocolchicine to 40% and that of the L-isomer to 35%.

Oxidation of desmethyl thiocolchicine by means of Javelle water under the same conditions as set forth in Example 2 yield the same compounds in slightly smaller yields.

By reduction of the two sulfoxides by means of sodium bisulfite under the same conditions as described above, there is obtained desmethyl thiocolchicine (Formula II, R=H; R1=-COCH3; R2=-CH3).

Example 4.Pnoduction of D- and L-sulfoxides of ethyl thiocolchiceine (Formulae Ia and 1b, R=CH3, R1=CO-CH3, R2=-C2H5) 1.5 g. of ethyl thiocolchiceine are dissolved in 30 cc. of chloroform, a 25% ethereal solution of perphthalic acid is added thereto in the cold, reaction is allowed to occur 6 until 1 mol of the peracid has been consumed, and the mixture'is then allowed to stand for 30 minutes at room temperature. The resulting liquid is poured into water, the aqueous layer is decanted and the solution in chloroform is washed with sodium bicarbonate and with water, dried, and finally evaporated to dryness. The residue is taken up in ethyl acetate. After addition of ether, 665 mg. (43%) of a crude product are obtained which are recrystallized from a mixture of 5 volumes of dimethyl formamide and 15 volumes of ether. The pure L-sulfoxide of ethyl thiocolchiceine melts at 230 C. with decomposition, [oc] =-574i10 (0.33% concentration in chloroform). This product is present in the form of yellow crystals which are insoluble in water, ether, petroleum ether, but are soluble in chloroform and dimethyl formamide, and which are sparingly soluble in ethyl acetate.

Analysis.-Found: C%,'62.0; H%, 6.1; 0%, 20.9; N%, 3.2; S%, 6.9. C23H2706NS (445.52) requires C%, 62.0; H%, 6.1; 0%, 21.6; N%, 3.1; S%, 7.2.

The mother liquors from which the L -sulfoxide of ethyl thiocolchiceine in the crude state has crystallized are evaporated to dryness. The residue is taken up in ethyl acetate and, after the addition of ether, 605 mg. (39%) of D-sulfoxide of crude ethyl thiocolchiceine are obtained; this compound is recrystallized from a mixture of 5 volumes of dimethyl formamide and 15 volumes of ether. The pure product melts at 240 C. with decom: position [a] :10 (0.33% concentration in chloroform). The product is present in the form of yellow prisms which are insoluble in water, ether, and petroleum ether, but soluble in chloroform and dimethyl formamide.

Analysis-Found: C%, 61.9; H%, 6.1; 0%, 21.9; N%,.3.0; S%,, 7.4. C23H2'1OsNS (445.52) requires C%, 62.0; H%, 6.1; 0%, 2 1.6; N%, 3.1; S%, 7.2.

By meansof chromatographic adsorption on neutral alumina of the mother liquors from which the D-sulfoxide of ethyl thiocolchiceine has crystallized, there is obtained the corresponding L-sulfoxide. The overall yield for the entire process is 86%.

Reduction of the D- and L-sulfoxides of ethyl thiocolchiceine in 30 volumes of methanol by means of 30 volumes of a 35% aqueous sodium 'bisulphite solution over a period of 45 minutes on a Water bath yields ethyl thiocolchiceine (Formula II, R=CH3, R1: CO-CHa, R2=C2H5).

Example 5.-Pr0duc'ti0n of D- and L-sulfoxides of ethyl thiocolchiceine 500 mg. of ethyl thiocolchiceine are dissolved in 3 cc. of acetic acid, 2 cc. of cold 30% hydrogen peroxide solution are added, and the resulting mixture is allowed to stand for two hours at room temperature. The reaction mixture is extracted with chloroform, washed, dried, and evaporated to dryness. The residue is treated as indicated in Example 4, and gives the identical D- and L- sulfoxides of ethyl thiocolchiceine.

Example 6.Pr0ducti0n of D- and L-sulfoxides of ethyl thiocolchiceine 1.5 g. of ethyl thiocolchiceine are dissolved in 30 cc. of chloroform, 20 cc. of a 3% solution of perbenzoic acid in chloroform are added thereto in the cold, and the reaction mixture is allowed to react until 1 mol. of the peracid has been consumed, and is then allowedto stand for 30 minutes at room temperature. The liquid reaction mixture is poured into water, the water is de canted, and the chloroform solution is washed, dried, and finally evaporated to dryness. The residue is treated as indicated in Example 4 and gives the identical D- and L-sulfoxides of ethyl thiocolchiceine.

Example 7,-Production of D and L-sulfoxides of ethyl thiocolchiceine 500 mg. of ethylthiocolchiceine are dissolved in 8 cc. of methanol, first 8 cc. of water and then 1 cc. of a hypochlorite solution (Javelle water) corresponding to one equivalent of active oxygen are added. An excess of solid sodium bicarbonate is introduced into this solution and a further cc. of Javelle water is added. The reaction mixture is heated for 30 minutes at 50 C., cooled, extracted with chloroform, and the chloroform extract is washed, dried, and evaporated to dryness. The residue is treated as indicated in Example 4 and the identical D- and L-sulfoxides of ethyl thiocolchiceine are obtained.

In place of thiocolchicine, ethyl thiocolchiceine, or desmethyl thiocolchicine used in the preceding examples, there can be used equimolecular amounts thereof of one of the following alkyl thiocolchiceine compounds. Otherwise, the procedure is the same as set forth in said examples.

Z-hydroxy ethyl thiocolchiceine,

N-desacetyl thiocolchicine,

N-desacetyl desmethyl thiocolchicine,

N-desacetyl-N-methyl thiocolchicine,

N-desacetyl-N-formyl thiocolchicine,

N desacetyl-N-formyl desmethyl thiocolchicine,

N-desacetyl-N-benzoyl thiocolchicine,

N-desacetyl-N-benzoyl desmethyl-O-benzoyl thiocolchicine,

N-desacetyl-N-benzoyl desmetyl thiocolchicine,

N-desacetyl-N-carbethoxy thiocolchicine,

N-desacetyl ethyl thiocolchiceine,

N-desacetyl-N-propionyl thiocolchicine,

N-desacetyl-Npropionyl desmethyl-O-propionyl thiocolchicine,

N-desacetyl-N-propionyl desmethyl ethyl thiocolchiceine,

Desmethyl ethyl thiocolchiceine,

Desmethyl isobutyl thiocolchiceine,

Isopropyl thiocolchiceine,

n-Desacetyl-N-p-toluoyl thiocolchicine,

n-Desacetyl-N-butyroyl thiocolchicine,

and the thiocolchiceine derivatives obtained according to our aforementioned application filed herewith.

In place of the oxidizing agents mentioned hereinbefore in the examples and in the specification there can be used other suitable oxidizing agents such as nitric acid, chromic acid, potassium permanganate in acetic acid at elevated temperature, other organic peracids, for instance, peracetic acid, lead tetraacetate, and others. A very suitable oxidizing agent is 30% hydrogen peroxide in acetic acid solution containing a certain amount of acetic acid anhydride.

It is understood, of course, that the solvents used in this process must be selected with care and that only such solvents are employed which are not afiected by the oxidizing agent. chloroform, for instance, is especially suitable when using hydrogen peroxide, perphthalic acid, perbenzoic acid, and other organic peracids as oxidizing agents. Oxidation by means of hydrogen peroxide is also satisfactorily effected in acetic acid and acetone while the preferred solvent for working with Javelle water is aqueous methanol.

The most preferred reaction temperature is at about 50 C. although lower and higher temperatures may also be employed. However, the temperature should not substantially exceed about 100 C. It is, of course, understood that reaction temperature and duration may vary considerably depending upon the starting material, the oxidizing agent, and the solvent employed.

As stated above, the new sulfoxides of alkyl thiocolchiceine compounds according to the present invention are used, for instance, in agriculture in the form of their solutions in suitable solvents or in mixture with suitable solid carrier materials. Concentrations between 0.02% and 0.002% have proved to be of great value in the treatment of seeds to produce polyploidism without, how-- ever, being limited thereto. Cultivated soil may also be;

sprayed with such solutions.

Of course, many changes and variations in the starting H3CO- NH.CO.CHI

o=i l 2. L-sulfoxide of methyl thiocolchiceine of the formula HaCQ- NH.CO.CH:

3. D-sulfoxide of desmethyl methyl thiocolchiceine of the formula H1O O NH,CO.CH;

| HaCO 4. L sulfoxide of desmethyl methyl thiocolchiceine of the formula moo- NH.CO.CH:

HaC (I) 5. D-sulfoxide of ethyl thiocolchiceine of the formula HsCO" .NH.CO.CH2

I CzHt 9 10 6. The sulfoxide compounds of the alkyl thioeolchiceine References Cited in the file of this patent series selected from the group consisting of D-sulfoxide Sautavy et Helm c Acta,"vo1 33, 1950, pages methyl thiocolchiceine, L-sulfoxide methyl thiocolchi- 1611,1514t 1627 ceine, D-sulfoxide 0f desmethyl methyl thiocolchicifle, Goldberg et al.: Cancer, vol. 3 (1950), pages 124 to L-sulfoxide of desmethyl methyl thiocolchiceine and D- 5 129.

lf xid of ethyl thimolchiceine, Lettre: Angewandte Chemie, vol. 63 (1951), pages 

6. THE SULFOXIDE COMPOUNDS OF THE ALKYL THIOCOLCHICEINE SERIES SELECTED FROM THE GROUP CONSISTING OF D-SULFOXIDE METHYL THIOCOLCHICEINE, L-SULFOXIDE METHYL THIOCOLCHICEINE, D-SULFOXIDE OF DESMETHYL METHYL THIOCOLCHICEINE, L-SULFOXIDE OF DESMETHYL METHYL THIOCOLCHICEINE AND DSULFOXIDE OF ETHYL THIOCOLCHICEINE. 